We are studying polypeptide-secreting and circadian oscillator neurons in tissue culture as dissociated cell systems from the opisthobranch mollusk Aplysia. The intact but isolated eye of Aplysia has a built-in neuronal circadian oscillator which can free-run for about two weeks in vitro, in darkness. Entrainment and temperature compensation of the circadian period are inherent mechanisms within the eye. We are dissociating the eye into individual cells in order to localize the ciradian oscillator. Light and electron-microscopic (both scanning and transmission) studies of such dissociated cells are being performed for identification purposes. Short-term intracellular and long-term extracellular electrical recordings are being used to identify neuron types involved in ultradian and circadian rhythms. Time-lapse motion picture studies of nuclear and cytoplasmic changes in these neurons over the 24-hour period are being obtained. Isolated photoreceptors can be studied electrophysologically for long periods and show stable depolarizing responses to light. The bag cell neurons synthesize and release a 4400 molecular weight polypeptide (ELH) which induces behavioral egg-laying. The special developmental, electrophysiological and biochemical properties of this peptidergic system are being studied in tissue culture where single neurons and small connected clusters can be examined under more rigorously controlled conditions. The mechanism of "afterdischarge" is being investigated in these neuroendocrine cells with particular emphasis on the role of cAMP and protein phosphorylation. Immunocytochemical studies are being conducted in the bag cells, in primary culture, with rabbit antibodies to purified ELH.